Anti-lactose antibodies

Ghose, A.C. Karush, F. (1973). The affinity and temporal variation of isoelectric fractions of rabbit anti-lactose antibody. Biochemistry 12, 2437-2443. 

Rodwell, J.D. Karush, F. (1980). Restriction in IgM expression-I. The VH regions of equine anti-lactose antibodies. Mol. Immunol. 17, 1553-1561. 

Maa et al. [3.84] used spray drying and spray freeze-drying (see Chapter 5, [5.13, 5.14]) to produce protein powders for inhalation from deoxyribonuclease (rhDNase) and anti-IgE monoclonal antibody (anti-IgE Mab) with lactose as carrier. Spray freezedrying produced light and porous protein particles with superior aerosol performance. 

That both components were antibodies was established by agar diffusion tests with these preparations and solutions of the glycan performed in the usual manner. The results of the agar diffusion tests are shown in Figure 3, right plate. The two preparations of antibodies have been designated as anti-galactose (anti-gal) antibodies and anti-lactose (anti-lac) antibodies and some of the properties of the two sets of antibodies are described in a later section. 

The homogeneity and molecular size of the purified antibody preparations were determined by ultracentrifiigation in a Spinco Model E centrifuge. The results of this analysis on anti-lactose antibodies are illustrated in the photographs of the ultracentrifuge patterns shown in Fig. (1). The... 

Fig. (1). Spinco Model E ultracentrifiigation pattern for anti-lactose antibodies.

Fig. (7). Affinity chromatography pattern for isolation of anti-lactose antibodies, n-p, non-proteins Ah, antibody.

Fig. (9). Inhibition of anti-lactose antibodies by increasing amounts of methyl P-lactoside and lactose (A) and by the agar diffusion method (B).

The specificity of the antibodies can be verified by inhibition tests using agar diffusion data [35], The results of such experiments with anti-lactose... 

Fig. (10). Agar diffusion of different proteins and a lipid conjugated with lactose against anti-lactose antibodies (well L) and against anti-BSA antibodies (Well BS) wells 1-6 contained Lac-poly, Lac-BSA, Lac-sphingosine, Lac-ORA, Lac-HGG and BSA Chemical modification of the antigen by periodate oxidation or borohydride reduction can effect an agar diffusion against anti-gum arabic antibodies (Se), GA=gum arabic Bl=blank.

Fig. (11). Isoelectric focusing pattern for anti-lactose antibodies composed of 13, 16 and 21 Isoforms.

Fig. (15). Agar diffusion (AX electrophoresis (B) and isoelectric focusing (C-F) of anti-lactose antibodies.

Gel electrophoresis yielded a broad band and indicated several protein components, Fig. (15B). It was necessary to establish whether the preparation contained several types of antibodies or only anti-lactose antibodies. The preparation was subjected to electrofocusing [39], Duplicate gels were made by electrofocusing. One gel was stained for protein and showed the presence of multiproteins, Fig. (15C). The unstained gel was examined by the coupled method and yielded a precipitin which was opposite each band in the stained gel, Fig. (15D-F). These results showed that the purified antibody preparation consisted of 13 protein species each with antibody activity directed at lactose-containing antigen. 

Fig. (16)l Liquid dectrofocused pattern (A) electrophorised in gels stained by protein reagent (B) and agar diffusion (C) of anti-lactose antibodies. 

Fig. (17). Gel electrophoresis (A) and sedimentation on density gradient ultra centrifugation (B) °f anti-lactose antibodies and dissociated antibodies. Antibody (Ab), reaction mixture (R), heavy chain (H), light chain (L). ...

Fig. (18). Isoelectric focusing of anti-lactose antibodies (Ab) and the Heavy (H) and the Light (L) chains.

Fig. 5.—Inhibition of anti-lactose antibodies reacting with an antigenic polysaccharide from a Group D Streptococcus in the presence or absence of inhibitors well A = anti-lactose antibodies well B = antibodies with lactose well C = antibodies with galactose wells 1-6 contain the antigen at decreasing concentrations from 20 to 1 p,g. (Reprinted from Journal of Immunological Methods, Volume 75, J. H. Pazur, and S. A. Kelly, pp. 107-116, copyright 1984 with kind permission of Elsevier Science—NL, Sara Burgerhartstraat 25, 1055 KV Amsterdam, The Netherlands.)...

Sepharose and elution with lactose (Fig. 23). The UV-absorbing fraction that was eluted with the lactose solution was collected and the protein precipitated by addition of an equal volume of saturated ammonium sulfate. On refrigeration of the sample overnight, a white precipitate formed. This precipitate was collected by centrifugation and redissolved in 0.2 mL of 0.02 M phosphate buffer of pH 7 in saline. The agar-diffusion test shown in the inset of Fig. 23 showed that high-titer antibodies were produced that formed a precipitin complex with the antigen. Several affinity runs were made. The antibody samples were combined and lyophilized. The yield of anti-lactose antibody was 50 mg from 20 mL of serum. 

Fig. 24.—A Hapten inhibition of the anti-lactose antibodies by lactose and galactose and derivatives. B Dissociation of anti-lactose antibodies into light and heavy chains followed by density-gradient centrifugation. (A. Reprinted with permission from Journal of Biological Chemistry, Volume 253, J. H. Pazur, K. L. Dreher, and L. S. Forsberg, pp. 1832-1837, copyright 1978 Journal of Biological Chemistry B, Reprinted with permission from Journal of Protein Chemistry, Volume 6. J. H. Pazur, M. E. Tay, B. A. Pazur, and F. J. Miskiel, pp. 387-399, copyright 1987 Journal of Protein Chemistry.)...

Fig. 25.—Gel electrophoresis and isoelectrofocusing of anti-lactose antibodies followed by agar diffusion. A, electrophoresis gel 1 stained with Coomassie Blue, gel 2 embedded in agar, area of precipitin formation (3), and solution of antigen (4). B, electrofocusing gel patterns (5-8) similar to series (1-4).

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